This proposal aims to explore and test a novel targeted oncolytic viro-cell therapy using the mesenchymal stem cells (MSCs) infected by harmless common cold virus, the respiratory syncytial virus (RSV) for treatment of lung cancers, which is the number one killer among cancers worldwide. Despite advances in the treatment and improvement in outcomes, the 5-year survival rates remain very poor (15% or less) for non?small cell lung cancers (NSCLC), which constitute ~85% of all lung cancer patients. Such poor survival rates combined with lung cancer heterogeneity and development of resistance to classical chemo- and radio-therapies, underscore the need for development of novel non-palliative therapeutic strategies. Though oncolytic virotherapies have emerged as a potential alternative to traditional chemo- and radio-therapies, their application to lung cancers remain limited. Also, inability to target oncolytic viruses to tumors in general remains a critical barrier and particularly for lung cancers remains a major unmet need. The motivation for the concept of targeted oncolytic viro-cell therapy against lung cancer comes from two major developments. First, MSCs, a cell-type generally known to have intrinsic tumor tropic properties, were found to be highly susceptible to RSV, with >90% infection rate, suggesting the use of RSV-infected MSCs for both tumor targeting and tumor killing. This potential was challenged by upregulation of indoleamine-2, 3-dioxygenase (IDO) by RSV-infected MSCs, which suppresses tumor immunity and thus aid in tumorigenesis. However, it was found that IDO- deficient hMSCs remain highly susceptible to RSV while alleviating the tumor suppressive effect. Second, nonstructural protein 1 (NS1) deficient (?NS1) RSV replicates with high viral titer in lung tumor cells, compared to the normal WI-38 diploid lung cells. Together these findings have led to the hypothesis that the ?NS1 RSV-infected IDO-deficient MSCs will retain tumor tropism and would lend themselves to develop a targeted oncolytic viro-cell therapy against lung cancers. This hypothesis will be tested in the following three specific aims. ! In aim #1, it is planned to develop and characterize a stable IDO-deficient hMSC cell line using the CRISPR method and examine the anti-tumor activity potential of wild type and engineered (?NS1) RSV-infected MSCs (RMSCs) in 3D multicell tumoroid cultures in vitro and biopsy-derived tumoroids ex vivo. In aim #2, it is proposed to assess the anti-cancer potential of RMSCs in vivo in immunocompetent syngeneic orthotopic model of lung cancer and evaluate their potential to modulate anti-tumor immunity. In aim #3 the effects of RMSC in modulating apoptosis induced by viral antigens will be investigated. Overall, the targeted RMSC therapy is expected to provide a highly innovative and effective approach for treatment of lung cancers. The PIs are uniquely positioned to conduct the proposed studies to develop a novel therapeutic approach for lung cancers, which may apply to other neoplastic diseases. All the required methods, reagents and collaborations are in place for the successful completion of this proposed program with highest clinical impact in the lung cancer field.